Proteins having a flavonoid 5-O-glycosyltransferase activity (5GT)

ABSTRACT

The present invention provides a gene that codes for a protein, wherein the protein is an anthocyanin 5-O-glucosyltransferase, which is an enzyme that transfers a glycoside to the 5 position of a flavonoid. The disclosed anthocyanin 5-O-glucosyltransferases were isolated from  Perilla fruescens, Verbena hybrida, Torenia hybrida , and  Petunia hybrida . Also disclosed are genes that code for a protein having a modified amino acid sequence relative to the above amino acid sequence and having activity that transfers a glycoside to the 5 position of a flavonoid, and a process for producing the above protein using said gene. This gene can be used to artificially alter the color of plants.

TECHNICAL FIELD

The present invention relates to a gene coding for a protein havingactivity that transfers a glycoside to the 5 position of a flavonoid,and a process utilizing that gene.

BACKGROUND ART

The flower industry strives to develop various new varieties. Changingthe color of a flower is one way of effectively breeding a new variety.A wide range of colors have been successfully produced for nearly allcommercial varieties using classical breeding methods. With thesemethods, however, since there are restrictions on the gene pool for eachspecies, it is rare for a single species to have a broad range ofcolored varieties.

Flower colors are based on two types of pigments, namely flavonoids andcarotinoids. Flavonoids contribute to color tones ranging from yellow tored and blue, while carotinoids contribute to color tones of orange oryellow. Flavonoid molecules that primarily contribute to flower colorare anthocyanins which are glycosides of cyanidin, delphinidin,petunidin, peonidin, malvidin and pelargonidin, and different anthocyanscause remarkable changes in flower color. Moreover, flower color is alsoaffected by auxiliary coloring by colorless flavonoids, metal complexformation, glucosylation, acylation, methylation and vacuolar pH(Forkmann, Plant Breeding, 106, 1, 1991).

The biosynthesis route of anthocyanins, which begins with phenylalanine,has been well understood (e.g., Plant Cell, 7, 1071-1083, 1995), andnearly all genes involved in the biosynthesis have been cloned. Forexample, among those genes thought to be involved in biosynthesis ofmalonylshisonin(3-0-(6-0-(p-cumaloyl)-β-D-glucosyl)-5-0-(6-0-malonyl-β-D-glucosyl)-cyanidin),which is an anthocyanin of Perilla, those genes for which homologueshave not yet been reported are only the flavonoid-3′-hydroxylase,UDP-glucose: anthocyanin (flavonoid) 5-0-glucosyl transferase(abbreviated as 5GT) and malonyl group transferase genes.

Among these, flavonoid-3′-hydroxylase is known to belong to thecytochrome P450 gene family (Plant Cell, 7, 1071-1083, 1995), andcytochrome P450 genes are surmised to demonstrate structural homology.

The hydroxyl group at the 3 position of flavonoid molecules is typicallymodified by glucose, and generally glucosylation and other modificationsby glycoside are considered to increase the stability and solubility ofanthocyanins (The Flavonoids, Chapman & Hall, 1994).

Genes coding for the UDP-glucose: anthocyanidin or flavonoid-3-glucosyltransferase (abbreviated as 3GT) that catalyze this reaction areobtained from numerous plants such as corn, barley, snapdragons andgentians, and their amino acid sequences mutually demonstratesignificant homology. For example, the homology between the 3GT aminoacid sequences of monocotyledonous corn and dicotyledoneous gentian is32%, that between the 3GT amino acid sequences of monocotyledonous cornand monocotyledonous barley is 73%, and that between the 3GT amino acidsequences of dicotyledonous gentian and dicotyledonous eggplant is 46%.

In addition, the gene coding for UDP-ramnose: anthocyanidin3-glucosidoramnosyl transferase (3RT) of petunias has also been cloned.

However, even though the hydroxyl group at the 5 position of theflavonoids of numerous plants is glucosylated, a gene for the enzyme(5GT) that catalyzes this reaction has yet to be obtained.

In addition, although there are examples of measuring the reaction bywhich glycoside is transferred to the 5 position of petunia and stockanthocyanins (Planta, 160, 341-347, 1984, Planta, 168, 586-591, 1986),these reports only describe the investigation of enzymologicalproperties using crude extracts or partially purified products of flowerpetals, and there are no examples of this enzyme being purified to itspure form. In addition, since glycosyltransferases are typicallybiochemically unstable, enzyme purification is difficult.

Although there are hardly any cases in which color tone is changed byaddition of glycoside to a flavonoid molecule, since aromatic acylgroups that have a significant effect on color tone are linked to aglucose molecule or ramnose molecule within an anthocyanin, regulationof the glycoside transfer reaction is important in terms of controllinganthocyanin biosynthesis, and ultimately in controlling flower color.Furthermore, as an example of changing flower color by regulating theexpression of glycosyltransferase gene, the reaction by petunia 3RT hasbeen controlled in transformed petunia to modify flower color.

Plant species, which can be transformed with a foreign gene, include,for example, roses, chrysanthemums, carnations, daisies, petunias,torenia, bellflowers, calanchoes, tulips and gladiolas.

DISCLOSURE OF THE INVENTION

The inventors of the present invention therefore sought to obtain a genethat codes for a protein having activity that transfers a glycoside tothe 5 position of a flavonoid, thereby leading to completion of thepresent invention.

For example, the 5 position hydroxyl group of the anthocyanins ofchrysanthemums and some of the anthocyanins of roses and carnations arenot glucosylated. The anthocyanin structure can be changed byintroducing the 5GT gene obtained by the present invention into theseplants.

In addition, although it is possible to change flower color andstabilize flavonoids by acylating flavonoids using the acyl grouptransferase gene described in International Publication No. WO96/25500,since the acyl group does not bond directly with the flavonoid, butrather bonds by way of a sugar, simply introducing an acyl grouptransferase gene alone is not sufficient for changing flower color andmay even cause the flavonoid not to become stable.

However, by introducing the 5GT gene in combination with an acyl grouptransferase gene, sugar is bounded to the 5 position of the flavonoidthereby further allowing the flavonoid to be acylated. This can beexpected to change the anthocyanin structure and cause the flower colorto become bluish.

In addition, if expression of 5GT gene of a plant in which the 5position of anthocyanin is glucosylated is suppressed with the antisensemethod or co-suppression method and so forth, transfer of glucoseresidue to 5 position can be inhibited. So that, flower color can bechanged. For example, suppressing 5GT activity in gentian or bellflowercan be expected to cause flower color to become reddish.

The inventors of the present invention isolated cDNA of 5GT fromPerilla, torenia, verbena and petunia plants using gene recombinationtechnology, and determined the nucleotide sequence of the structuralgene. Namely, the inventors of the present invention provide a DNAsequence that codes for 5GT present in the tissue that expressesanthocyanins in these plants. Moreover, since this enzyme transfersglycoside to the 5 position of anthocyanin pigment, it can be used tochange flower color and increase anthocyanin stability.

EMBODIMENT FOR CARRYING OUT THE INVENTION

The method of differential displacement, for example, can be used toobtain DNA that codes for the enzyme of the present invention. InPerilla (Perilla frutescens), for example, there are varieties thataccumulate anthocyanins (e.g., red forma) and those that do not (e.g.,green forma). By cloning DNA present in varieties that accumulateanthocyanins but not present in varieties that do not, it is possible toobtain the DNA that codes for the enzyme of the present invention.

More specifically, RNA is extracted from the leaves of red forma andgreen forma, and cDNA is synthesized in accordance with standardmethods. This is then separated by electrophoresis to isolate cDNApresent in the cDNA library of red forma but not present in the cDNAlibrary of green forma. Next, the red forma cDNA library is screenedusing the resulting cDNA as a probe to obtain the cDNA that codes forthe enzyme of the present invention.

Once cDNA that codes for the enzyme of the present invention is obtainedin the manner described above, this cDNA or its fragment is used as aprobe to screening the cDNA libraries of other plants. As a result, theDNA that codes for the enzyme of the present invention can be obtainedfrom those plants.

As an example of the screening, in the present invention, the DNA codingfor the enzyme of the present invention is cloned from Perilla by thedifferential display method (Example 1). Next, DNA that codes for theenzyme of the present invention is obtained from verbena by screening ofcDNAs from verbena (Verbena hebrida) using the cloned DNA of Example 1as a probe (Example 2). Moreover, DNA coding for the enzyme of thepresent invention is obtained from torenia in the same manner (Example3).

Then, it was confirmed that the proteins encoded in these DNAs have theenzymatic activity of the present invention.

Moreover, the DNA coding for the enzyme of the present invention wasobtained from petunia (Example 4).

Examples of the DNAs of the present invention include that which codesfor the amino acid sequence described in any one of SEQ ID NOs: 2, 4, 6,8, or 12. However, proteins having an amino acid sequence modified byaddition and/or deletion of one or more amino acids and/or substitutionsby one or more other amino acids are also known to maintain enzymaticactivity similar to the original protein. Thus, genes coding for aprotein that has an amino acid sequence modified by addition and/ordeletions of one or more amino acids and/or substitutions by one or moreother amino acids relative to the amino acid sequence described in anyone of SEQ ID NOs: 2, 4, 6, 8, or 12, and still maintains activity oftransferring a glycoside to the 5 position of a flavonoid, also belongto the present invention.

The present invention also relates to a gene coding for a protein whichgene hybridizes to a nucleotide sequence described in any one of SEQ IDNOs: 1, 3, 5, 7, or 11, or to a nucleotide sequence that codes for anamino acid sequence described therein or to their portions, for examplea portion coding for at least six amino acids of a consensus region,under conditions of 2 to 5×SSC, and for example, 5×SSC, and 50° C., andthat has activity of transferring a glycoside to the 5 position of aflavonoid. Furthermore, the optimum hybridization temperature variesaccording to the nucleotide sequence and its length, and it ispreferable that the hybridization temperature be lower the shorter thenucleotide sequence. For example, a temperature of 50° C. or lower ispreferable in the case of a nucleotide sequence (18 bases) coding forsix amino acids.

Although examples of genes selected by hybridization in this mannerinclude those which are naturally-occurring such as those derived fromplants, examples of which include a gene derived from verbena andtorenia, they may also be those derived from other plants, examples ofwhich include petunias, roses, carnations and hyacinths. In addition,genes selected by hybridization may also be cDNA or genomic DNA.

Moreover, the present invention also relates to a gene coding for aprotein having an amino acid sequence having homology of 30% or more,preferably 50% or more, for example 60% or 70% or more, and in somecases, 90% or more relative to an amino acid sequence of any of SEQ IDNOs: 2, 4, 6, 8, or 12, and having activity that transfers a glycosideto the 5 position of a flavonoid. Namely, as indicated in Examples, DNAcoding for the enzyme of the present invention demonstrates homology of20 to 30% in comparison with other glycosyltransferase genes. Thus, thepresent invention includes genes coding for a protein that havinghomology of 30% or more with an amino acid sequence described in any oneof SEQ ID NOs: 2, 4, 6, 8, or 12, and has glycosyltransferase activity.

In addition, as is clear from a comparison of the results of Examples 1through 4, the amino acid sequence of the enzyme of the presentinvention varies according to the species, with interspecies homologybeing 50% or more (see Examples 3 and 4), and for example 60 to 70% (seeExample 2), while the homology of the amino acid sequences of theenzymes derived from the same species is 90% or more (see Example 1).Thus, genes coding for a protein that has an amino acid sequence havinghomology of 50% or more, for example 60-70% or more, and in some cases,90% or more, relative to an amino acid sequence described in any one ofSEQ ID NOs: 2, 4, 6, 8, or 12, and maintains the glycosyltransferaseactivity of the present invention are included in the present invention.

As is described in detail in Examples, DNA having a native nucleotidesequence is obtained by, for example, screening of a cDNA library.

In addition, DNA coding for an enzyme having a modified amino acidsequence can be synthesized using ordinary site-specific mutagenesis andPCR based on the nucleotide sequence of a native DNA. For example, a DNAfragment containing a site at which a modification is desired to beintroduced is obtained by restriction enzyme digestion of cDNA orgenomic DNA obtained as described above. By using this as a template,site-specific mutagenesis or PCR is performed using a primer containingthe desired mutation to obtain a DNA fragment containing the desiredmodification. This is then ligated to DNA coding for another portion ofthe target enzyme.

Alternatively, in order to obtain DNA coding for an enzyme having ashortened amino acid sequence, for example, DNA coding for an amino acidsequence that is longer than the target amino acid sequence, for examplethat coding for the entire amino acid sequence, is digested by a desiredrestriction enzyme, and in the case the resulting DNA fragment does notcode for the entire target amino acid sequence, the deficient portionshould be supplemented by ligating synthetic DNA.

In addition, by expressing this clone using a gene expression system inE. coli or yeast and measuring enzyme activity, the resulting gene canbe confirmed to code for glycosyltransferase, and by clarifying thetranslation region of glycosyltransferase gene that transfers glycosideto the 5 position of a flavonoid, a gene is obtained that codes for theglycosyltransferase claimed in the present invention. Moreover, byexpressing said gene, the target transferase protein that transfers aglycoside to the 5 position of a flavonoid can be obtained.

Alternatively, the protein can be obtained by using antibody to an aminoacid sequence described in any one of SEQ ID NOs: 2, 4, 6, 8, or 12.

Thus, the present invention also relates to a recombinant vectorcontaining the above-mentioned DNA, and more particularly, to anexpression vector and a host transformed with the vector. Bothprokaryotes and eukaryotes can be used for the host. Examples ofprokaryotes that can be routinely used for the host include bacteria,for example, the genus Escherichia such as Escherichia coli, and thegenus Bacillus such as Bacillus subtilis.

Examples of eukaryotes that can be used include lower eukaryotes such aseucaryotic microorganisms including fungi such as yeast or mold.Examples of yeast includes the genus Saccharomyces such as Saccharomycescerevisiae, while examples of molds include the genus Aspergillus suchas Aspergillus oryzae and Aspergillus niger, as well as the genusPenicillium. Moreover, animal or plant cells can also be used, examplesof animal cells including mouse, hamster, monkey and human cell systems.Moreover, insect cells such as silkworm cells or adult silkwormsthemselves can be used as hosts.

The expression vectors of the present invention contain an expressioncontrol region, such as a promoter, terminator or an origin ofreplication, depending on the type of host in which they are to beintroduced. Examples of promoters of bacterial expression vectorsinclude conventionally used promoters such as trc promoter, tac promoterand lac promoter, while examples of yeast promoters includeglyceroaldehyde triphosphate dehydrogenase promoter and PH05 promoter.Examples of mold promoters include amylase and trpC. In addition,examples of promoters for animal cell hosts include viral promoters suchas SV40 early promoter and SV40 late promoter.

Preparation of expression vector can be performed in accordance withstandard methods using restriction enzyme, ligase and so forth. Inaddition, transformation of a host by an expression vector can also beperformed in accordance with standard methods.

In the process for producing the above-mentioned protein, a hosttransformed with the expression vector is cultured, cultivated or bred,the target protein can be recovered and purified from the resultingculture in accordance with standard methods, examples of which includefiltration, centrifugation, cell homogenation, gel filtrationchromatography and ion exchange chromatography.

Furthermore, although the present specification describes transferasesderived from Perilla, verbena, torenia and petunia wherein thetransferases that transfer glycoside to the 5 position of a flavonoid(which may be simply referred to as “glycosyltransferase” in the presentinvention), a gene that codes for said enzyme can be cloned, by entirelyor partially altering the purification method of said enzyme so as topurify a glycosyltransferase of another plant, and determining the aminoacid sequence of said enzyme. Moreover, by using cDNA of theglycosyltransferase derived from Perilla of the present invention as aprobe, cDNA of a different glycosyltransferase was able to be obtainedfrom Perilla, and cDNA of a different glycosyltransferase was able to beobtained from a different plant. Thus, other glycosyltransferase genescan be obtained by using a portion or the entirety of aglycosyltransferase gene.

In addition, as indicated in the present specification, by purifyingglycosyltransferase from Perilla, verbena, torenia and petunia to obtainantibody to said enzyme in accordance with standard methods, cDNA orchromosomal DNA produces protein which reacts with that antibody thatcan be cloned. Thus, the present invention is not limited to only genesof glycosyltransferases derived from Perilla, verbena, torenia andpetunia, but also relates to glycosyltransferase in the broad sense.

Moreover, the present invention also relates to a plant, its progeny ortheir tissue for which color has been adjusted by introduction ofglycosyltransferase gene, and their form may be that of cut flowers aswell.

In addition, UDP-glucose is an example of a glycoside donor in theglycoside transfer reaction of glycoside that include anthocyanin in thepresent specification.

EXAMPLES

The following provides a detailed explanation of the present inventionbased on Examples. Unless specified otherwise, the experimentalprocedure was performed in accordance with the methods described inMolecular Cloning (Cold Spring Harbor, 1989), New BiochemistryExperimental Manual (Kagaku Dojin, 1996) and International PatentLaid-Open Publication No. WO96/25500.

Example 1 Cloning of a Gene Specifically Expressed in Red Forma

(1) Differential Display

Perilla (Perilla frutescens) includes varieties that accumulateanthocyanins in their leaves (for example, red forma (Sakata-no-tane)),and varieties that do not accumulate anthocyanins (for example, blueforma (Sakata-no-tane)). The structure of the major anthocyanin isreported to be malonylshisonin(3-0-(6-0-(p-cumaloyl)-β-D-glucosyl)-5-0-(6-0-malonyl-β-D-glucosyl)-cyanidin)(Agri. Biol. Chem., 53:197-198, 1989).

Differential display is a method reported in Science, 257, 967-971(1992), and is used, for example, to obtain genes that are expressedtissue-specifically.

Total RNA was extracted from the leaves of the above-mentioned two typesof Perilla by the hot phenol method (Plant Molecular Biology Manual,Kluwer Academic Publishers, 1994, pp. D5/1-13). Poly A+RNA was purifiedfrom the resulting total RNA using an mRNA separator kit (Clonetech).0.9 μg of poly A+RNA were reverse-transcribed in 33 μl of reactionmixture using oligo-dT primer added an anchor (GenHunter, H-T11G,H-TL11A and H-T11C) to obtain single strand cDNA. Using this cDNA as atemplate, PCR was performed using the same oligo-dT primer added ananchor and synthetic primers (GenHunter, H-AP1 through 8) as primers.

The volume of the PCR reaction mixture was 20 μl, and it contained 2 μlof cDNA solution, 0.2 μM of any one of H-T11G, H-T11A or H-T11C primer,0.2 μM of any primer from H-AP1 through H-AP8, 0.12 μM dNTP, 5 or 10 μCiof [³²P]dCTP, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.01% Triton X-100,1.25 mM MgCl₂ and 1 unit of Taq polymerase. The reaction conditionscomprised holding the temperature at 72° C. for 20 seconds followed byrepeating the reaction for 40 cycles with one cycle comprising raisingthe temperature to 94° C. for 30 seconds, lowering to 40° C. for 2minutes and raising to 72° C. for 30 seconds, and then holding thetemperature at 72° C. for 5 minutes.

The DNA fragments amplified in this manner were separated by the samepolyacrylamide gel electrophoresis as used for DNA Sequencing. Afterdrying the gel, the gel was exposed to X-ray film. Among the resultingapproximately 2,600 bands, there were 36 bands observed only in the redforma as a result of comparing the two varieties. They were cut out ofthe dried gel and eluted into 100 μl of water. The eluted DNA wasprecipitated with ethanol and dissolved in 20 μl of water. Using a halfamount of each DNA as a template, the PCR reaction was performed asdescribed above, and amplified fragments were obtained for 33 of DNAfragments. Library screening and northern analysis were then performedusing these DNA fragments.

(2) Northern Analysis

Northern analysis was performed according to the method described belowusing the above 33 types of DNA probes. After separating poly A+RNAderived from red forma and green forma with formamide gel containing1.2% agarose, the poly A+RNA was transferred to a Nylon membrane. Thismembrane was hybridized with the above-mentioned DNA probes labeled with[³²P] for overnight at 65° C. in the presence of 5XSSPE, 5X Denhalt'ssolution, 0.5% SDS and 20 μg/ml of denatured salmon sperm DNA. Thehybridized membrane was washed at 65° C. in 1XSSPE and 0.1% SDS solutionand subjected to autoradiography. As a result, only five probes werespecifically expressed in red forma. These clones are predicted to begenes involved in the biosynthesis of anthocyanins.

(3) Screening of cDNA Library

A cDNA library with λgt10 as a vector was prepared using the poly A+RNAobtained from the leaves of red forma and the Complete Rapid CloningSystem λgt10 (Amersham). This cDNA library was screened with the fiveDNA fragments described above to obtain cDNA corresponding to eachfragment. Among these, a clone named 3R5 was obtained using a DNAfragment obtained by H-T11A and H-AP3 primers, and this clonedemonstrated homology of approximately 26% at the amino acid level withpreviously reported corn flavonoid-3-0-glucosyl transferase.

In addition, clones designated as 3R4 and 3R6 were obtained by libraryscreening using the same probes, and these demonstrated an extremelyhigh level of homology with 3R5. The complete nucleotide sequences anddeduced amino acid sequences of 3R4 and 3R6 are shown in SEQ ID NO: 1and SEQ ID NO: 3 of the Sequence Listing, respectively. In addition, thededuced amino acid sequences of the proteins encoded by 3R4 and 3R6demonstrated homology of 92%.

A clone designated as 8R6 was obtained using a DNA fragment obtained byH-T11G and H-AP8 primers, and this clone did not demonstrate significanthomology with any sequences reported so far. This sequence is shown inSEQ ID NO: 5 of the Sequence Listing. Although there is a strongpossibility that 8R6 is a gene involved in the biosynthesis ofanthocyanins, since its structure lacks homology with genes reported sofar, it is predicted to be a new gene involved in anthocyaninbiosynthesis.

In consideration of the anthocyanin structure in Perilla (the previouslymentioned malonylshisonin), it is predicted that this gene is a malonyltransferase. In order to verify this, this gene should be expressed inyeast and E. coli followed by reacting with anthocyanin and malonyl-CoAas substrates. Such an experiment can be carried out using, for example,the method described in International Publication No. WO96/25500.Malonyl transferase gene is useful in terms of artificially alteringanthocyanin structure.

(4) Expression of 3R4 cDNA in Yeast

An approximately 1.5 kb DNA fragment obtained by blunting the BstXIcleavaged site of p3R4 using T4 DNA polymerase (Takara Shuzo) and thencutting out at the BamHI cleavage site in the adapter, and anapproximately 8 kb DNA fragment obtained by blunting the EcoRI cleavedend of pYE22m and then digesting with BamHI were ligated to obtain aplasmid that was designated as pY3R4.

Furthermore, E. coli strain JM109 having pYE22m was named Escherichiacoli SBM335, and deposited at the National Institute of Bioscience andHuman-Technology Agency of Industrial Science and Technology as FERMBP-5435. In pY3R4, cDNA coding for glycosyltransferase has been ligateddownstream of the promoter for glyceroaldehyde triphosphatedehydrogenase lone of the constitutive yeast promoter, and transcriptionis controlled by this promoter.

Using pY3R4, yeast Saccharomyces cerevisiae G1315 (Ashikari, et al.,Appl. Microbiol. Biotechnol., 30, 515-520, 1989) was transformedaccording to the method of Ito, et al. (Ito, et al., J. Bacteriol., 153,163-168, 1983). The transformed yeast was selected according to recoveryof tryptophan synthesis ability. The resulting transformed strain wascultured for 24 hours at 30° C. with shaking in 10 ml of Burkholder'smedium (Burkholder, Amer. J. Bot., 30, 206-210) containing 1% casaminoacids.

In order to conduct a control experiment, yeast that spontaneouslyrecovered tryptophan synthesis ability was also cultured in the samemanner. After collecting the yeast, the cells were suspended insuspension buffer (100 mM phosphate buffer (pH 8.5), 0.1% (v/v)2-mercaptoethanol, 10 μM APMSF and 100 μM UDP-glucose) followed by theaddition of glass beads (Glass Beads, 425-600 microns Acid-Wash, Sigma)and vigorous shaking to crush the cells. The crushed cells were thencentrifuged for 20 minutes at 15,000 rpm and the supernatant was used asa crude enzyme solution for the measurement of enzyme activity describedbelow.

(5) Measurement of Enzymatic Activity

After allowing 50 μl of reaction mixture containing 20 μl of crudeenzyme solution (100 mM phosphate buffer (pH 8.5), 670 μMcyanidin-3-glucoside, 1 mM UDP-glucose) for 10 minutes at 30° C., 50 μlof 50% acetonitrile solution containing 0.1% TFA was added to stop thereaction. Supernatant obtained by centrifuging for 5 minutes at 15,000rpm was passed through a Samprep LCR4(T)-LC filter (Millipore) so as toremove impurities. This was then analyzed by high-performance liquidchromatography (HPLC). Analysis was performed using a reverse phasecolumn (Asahipak ODP-50, 4.6 mm diameter×250 mm, Showa Denko), themobile phase consisted of 0.5% TFA/H₂O for solution A and 0.5% TFA 50%CH₃CN for solution B. The flow rate was 0.6 ml/min. and the fractionswere eluted at a gradient of B20%→B100% (20 min) followed by holding atB100% for 5 minutes.

20 μl of reaction mixture was used for analysis. A520 nm, AUFS 0.5(Shimadzu SPD-10A) and a photodiode array detector (Shimadzu SPD-M6A) atan absorbance of 600-250 nm were used for detection. In the case ofreaction of yeast crude enzyme solution that expressed pY3R4, inaddition to the substrate cyanidin-3-glucoside (retention time: 17minutes), a new peak was observed at retention time of 14.5 minutes.Since it was not observed in the case of reaction of yeast crude enzymesolution of the control experiment, this new peak was considered to begenerated due to the activity of protein originated from pY3R4. As aresult of co-chromatography with cyanidin-3,5-diglucoside, the retentiontime of this peak coincided with that of cyanidin-3,5-diglucoside, andtheir absorption spectra were also identical to each other. Based onthese observations, 3R4 cDNA of Perilla was found to code for 5GT.

Example 2 Cloning of 5GT Gene of Verbena hybrida

(1) Preparation of cDNA Library

Petals were collected from Verbena variety Hanatemari violet (Suntory)and ground by a mortar and pestle in liquid nitrogen. RNA was extractedfrom the ground tissues according to a method using guanidinethiocyanate/cesium chloride, and poly A+RNA was obtained by the methodrecommended by the manufacturer using Oligotex (Takara Shuzo). Themethod using guanidine thiocyanate/cesium chloride was carried out inaccordance with the method described in detail in Methods in MolecularBiology, Vol. 2 (Humana Press Inc., 1984) by R. McGookin and Robert J.Slater, et al.

Using the resulting poly A+RNA as a template, double-stranded cDNA wassynthesized using the ZAP-cDNA synthesis kit (Stratagene), then, a cDNAlibrary was prepared using the Uni-ZAP XR Cloning Kit (Stratagene)according to the method recommended by the manufacturer.

(2) Cloning of 5GT cDNA

The λ phage library obtained as described above was screened in thefollowing manner using the p3R4 cDNA of Perilla as a probe. The filterswere maintained at 42° C. for 1 hour in hybridization buffer (5XSSC, 30%formamide, 50 mM sodium phosphate buffer (pH 7.0), 3% SDS 2% blockingreagent (Boehringer), 0.1% lauroylsarcosine, 80 μg/ml of salmon spermDNA). DIG-labeled Perilla 5GT cDNA, p3R4 cDNA, fragment was added to thehybridization solution and the filters were incubated for further 16hours.

After washing the filters with washing solution (5X SSC 50° C., 1% SDS),the positive clones labeled with anti-DIG-alkaline phosphate wereimmunologically detected using 5-bromo-4-chloro-3-indolylphosphate andnitro blue tetrazolium salt according to the method described by themanufacturer (Boehringer).

As a result, seven positive clones were obtained. These cDNA wereexcised on plasmid pBluescript SK using the method recommended byStratagene. When the lengths of the cDNA were investigated by agarosegel electrophoresis, insertion of a maximum length of 2.0 kb wasobserved.

(3) Determination of Nucleotide Sequence

Plasmids were extracted from the resulting clones, and the nucleotidesequences near the 3′ and 5′ ends of the cDNA were determined accordingto the dideoxy sequence method using fluorescent reagent as recommendedby Perkin-Elmer with the ABI 373A sequencer (Perkin-Elmer). As a result,five of the seven clones had mutually same nucleotide sequences althoughthe lengths of the cDNA were different. The entire nucleotide sequenceof pSHGT8 was determined. Determination of nucleotide sequences wasperformed as described above by either using the Kilo-Sequence DeletionKit (Takara Shuzo) to obtain a series of deleted cDNA clones, or byusing an oligoprimer specific for the internal sequence of pSHGT8.

(4) Comparison of the Nucleotide Sequence and the Amino Acid Sequence

The cDNA inserted into pSHGT8 had the length of 2062 bp, and included anopen reading frame (ORF) consisting of 1386 bp in length (including astop codon). This sequence is shown in SEQ ID NO: 5. The amino acidsequence of this ORF had homology of 68% with the amino acid sequence ofPerilla 5GT encoded by p3R4, and homology of 64% with that encoded byp3R6. In addition, it also had homology of 22 to 25% with the 3GTs ofmonocotyledonous and dicotyledoneous plants, and homology of 21% withpetunia 3RT.

(5) Expression in Yeast and Measurement of Enzymatic Activity

An approximately 2.0 kb DNA fragment obtained by digesting pSHGT8 withBamHI/XhoI, and an approximately 8 kb DNA fragment obtained by digestingpYE22m with BamHI/SalI were ligated, and the resulting plasmid wasdesignated as pYHGT8. pYHGT8 was expressed in yeast cells in the samemanner as Example 1, and the enzymatic activity of the protein encodedby pSHGT8 was measured. As a result, in the reaction mixture containingthe crude enzyme solution of yeast transformed with pYHGT8, a productwas obtained that coincided with cyanidin-3,5-diglucoside in bothretention time and absorption spectrum. Based on this observation, thepSHGT8 cDNA of Verbena was determined to code for 5GT.

Example 3 Cloning of Trenia 5GT Gene

(1) Preparation of cDNA Library

Petals were collected from torenia variety Summer Wave Blue (Suntory)and ground in a mortar and pestle in liquid nitrogen. RNA was extractedfrom the ground tissues according to a method using guanidinethiocyanate/cesium chloride, and poly A+RNA was obtained by the methodrecommended by the manufacturer using Oligotex (Takara Shuzo). Themethod using guanidine thiocyanate/cesium chloride was carried out inaccordance with the method described in detail in Methods in MolecularBiology, Vol. 2 (Humana Press Inc., 1984) by R. McGookin and Robert J.Slater, et al.

Using the resulting poly A+RNA as a template, double-strand cDNA wassynthesized using the ZAP-cDNA synthesis kit of Strategene, then, a cDNAlibrary was prepared using the Uni-ZAP XR Cloning Kit (Stratagene)according to the method recommended by the manufacturer.

(2) Cloning of 5GT cDNA

The λ phage library obtained as described above was screened in the samemanner as Example 2 using the p3R4 cDNA of Perilla as a probe. As aresult, eight positive clones were obtained. After excision of the cDNAon plasmid pBluescript SK, the lengths of the cDNA were investigated byagarose gel electrophoresis, which revealed that a maximum length ofinsertion was 1.6 kb.

(3) Determination of Nucleotide Sequence

Plasmids were extracted from the resulting clones, and the nucleotidesequences near both 5′ and 3′ ends were determined in the same manner asExample 2. As a result, six of the eight clones were considered to havemutually same nucleotide sequences although the lengths of the cDNA weredifferent. The entire nucleotide sequence of pSTGT5 cDNA was determined.

(4) Comparison of the Nucleotide Sequence and the Amino Acid Sequence

The cDNA encoded in pSTGT5 was of 1671 bp in length, and included anopen reading frame (ORF) consisting of 1437 bp in length (including astop codon). This sequence is shown in SEQ ID NO: 7. The amino acidsequence of this ORF had homology of 58% with the amino acid sequence ofPerilla 5GT encoded by p3R4, homology of 57% with that encoded by p3R6,and homology of 57% with that encoded by Verbena pSHGT8. In addition, italso had homology of 19 to 23% with the 3GT of monocotyledonous anddicotyledoneous plants, and homology of 20% with petunia 3RT.

(5) Expression of 5GT Gene in Yeast

An approximately 1.6 kb DNA fragment obtained by digesting pSTGT5 withSmaI/KpnI, and an approximately 8 kb DNA fragment obtained by bluntingthe EcoRI-digested site of pYE22m and then digesting with KpnI wereligated, and the resulting plasmid was designated as pYTGT5. pYTGT5 wasexpressed in yeast cells in the same manner as Example 1, and theenzymatic activity of the protein encoded by pSTGT5 was measured. As aresult, in the reaction mixture containing the crude enzyme solution ofyeast transformed with pYTGT5, a product was obtained that coincidedwith cyanidin-3,5-diglucoside in both retention time and absorptionspectrum. Based on this observation, the pSTGT5 cDNA of Torenia wasdetermined to code for 5GT.

Example 4 Cloning of Petunia 5GT Gene

(1) Preparation of cDNA Library

A cDNA library was prepared by RNA extracted from petals of the Petuniavariety Old Glory Blue in the manner described in detail by T. Holton,et al. (Plant Journal, 1993 4: 1003-1010)

(2) Cloning of 5GT cDNA

The cDNA library was screened in the same manner as Example 2 using themixture of 5GT cDNAs of Perilla, torenia and verbena obtained in themanner described above as probes. As a result, four positive cDNA cloneswere obtained and excised on plasmid pBluescript SK. The lengths of thecDNA were investigated by agarose gel electrophoresis, cDNA of a maximumlength of 2.0 kb was observed.

(3) Determination of the Nucleotide Sequence

Plasmids were extracted from the resulting clones, and the nucleotidesequence near the 5′ end was determined in the same manner as Example 2.As a result, two of the four clones, pSPGT1, were appeared to code anamino acid sequence with a high degree of homology with those of 5GTfrom Perilla, torenia and verbena obtained thus far. Therefore, theentire nucleotide sequence of pSPGT1 was determined.

(4) Comparison of the Nucleotide Sequence and the Amino Acid Sequence

The pSPGT1 cDNA was 2015 bp in length, and included an open readingframe (ORF) consisting of 1407 bp (including a stop codon). Thissequence is shown in SEQ ID NO: 11. The amino acid sequence of this ORFhad homology of 57% with that of 5GT encoded by p3R4 of Perilla,homology of 54% with that encoded by p3R6, 55% with that encoded bypSHGT8 of verbena, and 51% of that encoded by pTGT5 of torenia. Inaddition, it also had homology of 20 to 29% with the 3GT ofmonocotyledonous and dicotyledoneous plants, and homology of 20% withpetunia 3RT. Based on this observation, pSPGT1 cDNA obtained frompetunia is considered to code for 5GT.

INDUSTRIAL APPLICABILITY

As has been described above, cDNA coding for enzymes that transfer aglycoside to the 5 position of a flavonoid originating in Perilla,verbena, torenia and petunia were cloned and their nucleotide sequenceswere determined. In addition, the isolated cDNAs were clearly shown tocode for 5GT by the enzymatic activity of their protein expressed inyeast. Introducing of these cDNAs into a suitable plant expressionvector and transferring the resulting expression constructs into a plantmakes it possible to provide, increase or decrease 5GT activity in thetransformed plant, which leads to regulation of flower color. Inaddition, by using this enzyme, the structure of anthocyans can bealtered or more stable anthocyans can be synthesized either in plants orin vitro.

1. An isolated protein having an activity that transfers a glycoside tothe 5 position of a flavonoid, wherein said protein comprises a sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, and
 8. 2. Theisolated protein of claim 1, wherein said protein comprises the sequenceof SEQ ID NO:
 2. 3. The isolated protein of claim 1, wherein saidprotein comprises the sequence of SEQ ID NO:
 4. 4. The isolated proteinof claim 1, wherein said protein comprises the sequence of SEQ ID NO: 6.5. The isolated protein of claim 1, wherein said protein comprises thesequence of SEQ ID NO: 8.